One-carbon metabolism is a critical pathway in cancer epigenetics because it directs the methylation of DNA and RNA and is involved in DNA synthesis and repair. There is no good systemic marker of one-carbon metabolism status. Abnormal one-carbon metabolism may lead to genomic (global) hypomethylation in systemic blood DNA, which may predispose the development of neoplasia; if so, genomic methylation status in blood DNA may serve as a good systemic marker of one-carbon metabolism status. Several dietary factors including folate, alcohol, methionine, riboflavin, vitamins B6 and B12, choline, and betaine mediate or facilitate one-carbon metabolism pathway. We currently do not know the optimum balance of these multiple dietary factors to achieve maximum function in the pathway and to minimize cancer risk. Therefore, we propose to evaluate pre-diagnostic genomic methylation of leukocyte DNA in relation to colorectal cancer risk in large prospective studies and expect that individuals with reduced levels of genomic DNA methylation are at increased risk of colorectal cancer. We also propose to elucidate the associations between major plasma components of one-carbon metabolism (total folate, unmetabolized folic acid, 5-methyl-tetrahydrofolate, vitamins B6 and B12, cysteine, homocysteine, and methylene-tetrahydrofolate reductase gene) and genomic DNA methylation, to determine the role of each component in genomic DNA methylation and further validate the biological relevance of genomic methylation assessment in blood DNA. We finally propose to identify dietary predictors of genomic DNA hypomethylation, create a 'dietary methyl score', and examine the score in relation to colorectal adenoma/cancer risks. We hypothesize that the 'dietary methyl score' predicts risks of colorectal adenoma/cancer more strongly than the individual dietary predictors. We will also evaluate 'dietary methyl score' in relation to several molecular and epigenetic subtypes of colorectal cancer including tumors with LINE-1 hypomethylation, CpG island methylator phenotype (CIMP)-high, or microsatellite instability (MSI)- high. This application will take advantage of two large ongoing prospective follow-up studies of women and men with 666/1332 colorectal cancer cases/controls with pre-diagnostic blood samples for genomic methylation status of leukocyte DNA. We also expect to include 6,025 colorectal adenoma and 2,794 colorectal cancer cases to evaluate the relationships with 'dietary methyl score'. With several previously assessed plasma components of one-carbon metabolism and molecular subtypes of colorectal tumor, we are uniquely positioned to address these issues in an extremely time- and cost-effective manner. Our work will elucidate a new insight into how dietary factors and one-carbon metabolism affects the epigenetics of cancer in humans. Genomic methylation in leukocyte DNA can potentially serve as a diagnostic tool or target for cancer prevention because it is potentially modifiable. Dietary methyl score will help produce practical dietary guidelines for cancer prevention.